The detection of small amounts of peroxidatively active substances, such as occult blood, hemoglobin, myoglobin, leukocytes, bacteria, etc., in body fluids and in body excreta has long been recognized as an invaluable aid to the medical practitioner in the diagnosis of many abnormal conditions. for example, blood is found in the gastric contents, in vomitus, in urine, and in feces in conditions often associated with erosion of the gastric and intestinal mucous membranes. In the urine, the presence of these peroxidatively active substances may be indicative of such abnormal conditions as typhus, scurvy, purpura, pyemia, nephritis, third degree burns, carcinogenic conditions, disease and infection of the urinary system, hemolytic toxins and post-cardiac infarct. Since peroxidatively active substances are usually not present in the body fluids or excreta in macroscopic amounts, it is often difficult to rapidly detect the presence of these substances by clinical methods alone, even by microscopic examination. Therefore, it is considered highly desirable to provide a sensitive, rapid and reliable test for these substances.
Various procedures, compositions and devices are described in the literature for the detection of occult peroxidatively active substances. For example, Kamlet in U.S. Pat. No. 2,290,436; Nicholls and Fonner in U.S. Pat. No 2,799,660; Fonner in U.S. Pat. No. 2,838,377; and Adams and Peterson in U.S. Pat. Nos. 3,012,976, 3,092,463, and 3,092,464, all assigned to the instant assignee, illustrate several test compositions which have been supplied to meet the need for a simple, reliable test for occult blood. These test compositions are based on the peroxidative or catalytic activity of the prosthetic groups present in blood. The peroxidatively active substances identified in hemoglobin belong to the general class of hemoproteins, conjugate proteins, all of which have the same prosthetic groups, iron protoporphyrin or heme. This prosthetic group has the ability to catalyze the transfer of oxygen from an oxygen source to an acceptor which in turn becomes oxidized. If the acceptor is a dye precursor, colorless until it becomes oxidized and colored in its oxidized form, then the presence of a peroxidatively acitvie substance is indicated by color formation. The rapidity of the color change and the depth or intensity of the color when compared to a set of standards is then a means for the quantitative estimation of the blood present.
Although the above mentioned compositions provide a rapid means for the detection of occult blood, the compositions are relatively insensitive to expecially minute quantities of peroxidatively active substances corresponding to blood dilutions of less than about 1:20,000, i.e., about 200 to 300 intact red blood cells per microliter of sample (RBC.mu./ l). It has been determined by Adams et al. in U.S. Pat. No. 3,290,117 that the sensitivity of these occult blood test compositions can be markedly imporved and potentiated by the addition of quinoline or certain quinoline derivatives such as quinine. With the addition of these quinoline derivatives to the prior art occult blood compositions it is now possible to detect 5 to 50 RBC.mu./ l. of sample which corresponds to a blood dilution as low as 1:1,000,000. Another approach, disclosed in U.S. Pat. No. 3,853,472, reports the use of fused polycyclic derivatives of quinoline as potentiating or activating agents with similar sensitivities.
Since the sensitivity of the prior art occult blood tests is of such great importance, it is essential that these tests not only be highly sensitive to peroxidatively active substances, but also be stable, ie., they must retain their sensitivity. Unfortunately, except for those improved compositions containing quinine or certain other quinoline derivatives, many of the highly sensitive prior art compositions are unstable due to the volatility of the added potentiators at room temperatures or temperatures slightly above. Furthermore, virtually all of the prior art compositions utilize potentiators which are water insoluble and require suspension in organic solvents prior to incorporation into a test composition. For instance, in commercial practice, the known potentiators must be suspended in an organic solvent solution containing the indicators. As a result, the indicators are often rapidly discolored in the presence of these potentiators and must be discarded. It would therefore be highly desirable to provide test compositions which are not only rapid and highly sensitive but are also capable of retaining their sensitivity without discoloring the indicator system.